The invention relates to an improved solid phase immunological (preferably radioimmunological) assay method for the quantitative determination of antigens (Ag), e.g., hormones such as insulin, drugs such as digoxin, steroids such as the estrogens, etc. in a biological fluid, e.g., biological fluids from the body of an animal, such as blood serum, plasma, urine, gland extracts or gastric juices.
It also relates to an improved method for making as an article of manufacture an improved solid phase antibody (Ab) for use as such in carrying out said immunological assay method.
It also relates to a kit which includes tubes containing the improved solid phase antibody, a vial containing radioactive labelled antigen specific to the antibody, a vial containing buffer solutions to use clinically for assay of antigen and vials containing standard antigen amounts for preparation of a standard calibration curve, all packaged in a compact container.
Antigens (Ag) are substances which are capable by themselves or in combination with other substances of inducing the formation of specific antibodies (Ab) against themselves in an organism, e.g., in the body of an animal, into which they are introduced or in which they are formed to thereby neutralize the effect of the antigens.
Antibodies (Ab) are usually blood serum proteins of the globulin fraction, specific ones of which are formed in response to the introduction into the organism of specific antigens and which usually have substantially greater molecular weights than their antigens. The antibody neutralizes the effect of its antigen by binding the antigen to itself. The antibody (Ab) for a specific antigen (Ag) may be prepared by injecting the Ag into a rabbit or other animal. The animal produces the antibody to the antigen and the animal's blood can be processed (basically removal of red blood cells) to produce the antiserum containing the antibody.
In radioimmunological assay methods, which do not employ solid phase antibody, reaction (incubation) of solutions of non-labelled and labelled antigen Ag with solutions of antibody Ab forms a soluble Ab-Ag complex (the antigen becomes bound to the antibody) of relatively large molecular weight and size. Typically, porous solid materials such as charcoal are used onto which is adsorbed the labelled and non-labelled antigen of relatively small molecular weight and size, but not the Ab-Ag complex of larger molecular weight and size. By removing the supernatant from the charcoal and measuring the radioactivity of the charcoal containing unbound labelled and unlabelled antigen, or the supernatant containing bound unlabelled antigen and labelled antigen, quantitative measurement of the antigen in the biological fluid being tested can be achieved.
The use of solid phase antibody, e.g., as a solid layer, to react with the antigens has certain obvious advantages over the aforesaid non-solid phase techniques.
Since the present invention is directed to radioimmulological assay techniques employing solid phase antibodies, the discussion will be directed principally to these techniques.
Such techniques may be categorized into two classes: one is called IRMA, in particular, 2-site IRMA, and the other RIA.
Both solid phase antibody techniques are characterized by the pretreatment of a solid phase substrate surface, usually a polymeric tube or disc capable of adsorbing antibodies, with a solution of a specific antibody (Ab) for the antigen (Ag) to be assayed to effect adsorption or binding of the antibody as a solid phase layer to the substrate surface, to thereby achieve a solid phase antibody (Ab) surface. In effect, the Ab is precipitated out of solution onto the substrate as a solid phase Ab layer. The resulting solid phase antibody (Ab) is utilized in the quantitative determination of its antigen.
The IRMA technique utilizes radioactively labelled antibody whereas the RIA technique utilizes radioactively labelled antigen.
In the 2-site IRMA technique non-labelled antigen (Ag), in solution in the biological fluid being assayed, is reacted (incubated) with the solid phase antibody (Ab) to bind the Ag as a solid phase to the solid phase Ab followed by reaction (incubation) of the resulting solid phase antigen with radioactively labelled antibody (Ab*) whereby the Ab* becomes bound in solid phase to the solid phase antigen.
In the RIA technique, the radioactively labelled antigen (Ag*) and unlabelled antigen Ag are reacted or incubated with the solid phase Ab to form solid phase labelled and unlabelled antigen bound to the solid phase Ab.
These IRMA systems are discussed in Miles et al, "Properties of Two-Site Immunoradiometric (Labelled Antibody) Assay Systems", IAEA, 149, (1974), incorporated herein by reference.
Solid phase RIA systems are disclosed in U.S. Pat. Nos. 3,790,663, 3,646,346 and 3,826,619 as well as Catt et al, Science 158, 1570 (1967), all incorporated herein by reference.
All of the aforesaid solid phase radioimmunological techniques have disadvantages both in manufacture of the solid phase antibody and in clinical assay.
A common disadvantage is that relatively large concentrations and amounts of Ab are required to form the solid phase Ab. Specific antibodies Ab for specific antigens are often difficult to obtain continuously and consistently, particularly in the case of those antigens which are poor immunogens. For example, out of 100 rabbits injected with a specific antigen, a large number may not build up any specific Ab for that antigen at all and others may build up only a small amount. Accordingly, the supply of specific antibody is erratic, relatively expensive and sometimes tight. Therefore, there has been a long felt need for a method of making solid phase Ab utilizing substantially smaller concentrations and amounts of Ab.
The IRMA techniques require even more Ab than the RIA techniques because they employ radioactively labelled antibody Ab*. The IRMA procedure is also sensitive to serum effects during assay and is beset by the potential of antigen exchange via dissociation from the solid phase, i.e., during the reaction of the Ab* solution with the solid phase Ab-Ag, the previously deposited solid state Ag may dissociate from the solid phase. Such exchange would limit assay dose-response, particularly at high antigen concentrations.
Relatively large concentrations and amounts of Ab are required to form the solid phase Ab layer in present solid phase procedures in order to insure consistently adequate uniform and reproducible adsorption or deposit of Ab on the substrate to adequately and uniformly and consistently sensitize the solid phase substrate for uniform consistent and reproducible assay results. Different surface effects of the substrate tend to cause non-reproducible and non-uniform binding patterns and quantities (of the antibody to the substrate and hence of the antigen to the antibody) which effects the reproducability of assay results. More specifically, a relatively uniform and adequate amount of Ab must be consistently adsorbed on the substrate in order to achieve consistently adequate uniform and reproducible results and such uniformity is difficult to consistently achieve because of the different surface effects of the substrate.
Cocola et al in "New Radioimmunoassay Technique Using Second Antibody to Solid Phase Applied to Assays of Human Chorionic Somatomammotropin" Jour. Nucl. Biol. Med. 17, 174 (1973) discloses a procedure wherein solid phase anti-antibody Ab.sup.2, i.e., an anti-body built up in an animal against the serum of another animal, is adsorbed from solution as a solid phase on the substrate followed by contact of the resulting solid phase Ab.sup.2 with a solution of Ab and Ag to which a radioactive labelled antigen (Ag*) is subsequently added while still in contact with the solid phase Ab.sup.2. According to Cocola, the Ab, Ag and Ag* form soluble complexes Ab-Ag-Ag* which become bound as a solid phase to the solid phase Ab.sup.2.
The anti-antibody Ab.sup.2 is usually obtained immunologically by injecting the blood plasma or serum of one animal (e.g. a rabbit) into another animal, e.g., a sheep or goat, which builds up antibodies Ab.sup.2 against the serum of the rabbit. These anti-antibodies may be recovered from the sheep's blood as anti-antiserum containing the Ab.sup.2.
The term titre in the art means that amount of Ab which will result in about 50% binding of labelled antigen to Ab.
As aforesaid, antiserum containing Ab is obtained from the blood by separating out the red blood cells usually by centrifugation leaving the serum containing the antibody. This antiserum which may be further fractioned, is diluted with aqueous diluent (usually saline or buffer solutions) to various dilutions. A dilution of 1:50000 for example, means that the antiserum has been diluted to a ratio of 1 ml antiserum to 49,999 ml of diluent. A titre of 1:50000 means that dilution of antiserum is required to achieve about 50% binding of labelled antigen to Ab.
Cocola used an Ab.sup.2 titre or 1:10 to 1:50 and an Ab titre of 1:64000. Due to the comparative slowness of the immunogenic reaction (incubation) between the solid phase second antibody Ab.sup.2 and the mixture in solution of Ab, Ag and Ag*, Cocola's method requires an 18-24 hour clinical incubation period. This lengthy clinical incubation substantially limits the use of such technique in clinical applications.
The aforesaid Miles publication discloses in the IRMA technique, using radioactive labelled antibody Ab*, that the Ab, before reacting with Ag followed by reaction with Ab*, may be bound to the substrate surface by intermediate immunoglobulin arms either in the form of non-immune guinea-pig immunoglobulin (GP, IgG), as such, or as a rabbit-anti (GP IgG) in order to minimize serum effects during assay and improve dose response, his conclusion being that at least two arms are required to achieve this. This technique undesirably requires two clinical incubations (Ab-Ag and Ag-Ab*) and also a relatively large amount of Ab since it requires labelled Ab during assay in addition to the previously produced Ab.